The Vascular Endothelial Growth Factor (VEGF) family of growth factors and their receptors are essential regulators of angiogenesis and vascular permeability. The VEGF family comprises VEGF-A, PIGF (placenta growth factor), VEGF-B, VEGF-C, VEGF-E and snake venom VEGF and each is thought to have a distinct role in vascular patterning and vessel development. Due to alternative splicing of mRNA transcribed from a single 8-exon gene, VEGF-A has at least 9 subtypes (isoforms) identified by the number of amino acids remaining after signal peptide cleavage. For example, in humans the most prominent isoform is VEGF165, which exists in equilibrium between a soluble and cell associated form. Longer isoforms (VEGF183, VEGF189 & VEGF206) possess C-terminal regions that are highly positively charged and mediate association with cell surface glycans and heparin that modulates their bioavailability. All VEGF-A isoforms form homodimers with the association occurring via a core of approximately 110 N-terminal residues that constitutes the receptor-binding VEGF fragment. Under normal circumstances, and in the centre of solid tumours, expression of VEGF is principally mediated by hypoxic conditions, signifying a shortage of vascular supply. The hypoxia causes dimerization of the hypoxia inducible factor HIF-1α with the constitutively expressed HIF-1α, forming a transcription factor that binds to hypoxic response elements in the promoter region of the VEGF gene. Under normoxia, the HIF-1α protein undergoes ubiquitin-mediated degradation as a consequence of multiple proline hydroxylation events. Other tumour-associated VEGF up-regulation occurs due to activation via oncogene pathways (i.e. ras) via inflammatory cytokines & growth factors as well as by mechanical forces.
The active VEGF homodimer is bound at the cell surface by receptors of the VEGFR family. The principal vascular endothelium associated receptors for VEGF-A are VEGFR1 (Flt1) and VEGFR2 (Flk-2; KDR). Both receptors are members of the tyrosine kinase family and require ligand-mediated dimerization for activation. Upon dimerization the kinase domains undergo autophosphorylation, although the extent of the kinase activity in VEGFR2 is greater than that in VEGFR1. It has been demonstrated that the angiogenic signalling of VEGF is mediated largely through VEGFR2, although the affinity of VEGF is approximately 3-fold greater for VEGFR1 (KD˜30 pM compared with 100 pM for VEGFR2). This has led to the proposal that VEGFR1 principally acts as a decoy receptor to sequester VEGF and moderate the extent of VEGFR2 activation. Although VEGFR1 expression is associated with some tumours, its principal role appears to be during embryonic development & organogenesis. VEGF-A165 is also bound by the neuropilin receptors NRP1 & NRP2. Although these receptors lack TK domains, they are believed to acts as co-receptors for VEGFR2 and augment signalling by transferring the VEGF to the VEGFR2.
Numerous studies have helped confirm VEGF-A as a key factor in tumour angiogenesis. For example VEGF-A is expressed in most tumours and in tumour associated stroma. In the absence of a well developed and expanding vasculature system to support growth, tumour cells become necrotic and apoptotic thereby imposing a limit to the increase in tumour volume (of the order 1 mm3) that can result from continuous cell proliferation. The expression of VEGF-A is highest in hypoxic tumour cells adjacent to necrotic areas indicating that the induction of VEGF-A by hypoxia in growing tumours can change the balance of activators and inhibitors of angiogenesis, leading to the growth of new blood vessels in the tumour. Consistent with this hypothesis, a number of approaches, including small-molecular weight tyrosine kinase inhibitors, monoclonal antibodies, antisense oligonucleotides etc., that inhibit or capture either VEGF-A or block its signalling receptor, VEGFR-2, have been developed as therapeutic agents.
VEGF-A has also been implicated in a number of ocular diseases, such as age-related macular degeneration (AMD), wet AMD, geographic atrophy, diabetic retinopathy, retinal vein-occlusive diseases, diabetic macular oedema and corneal vascularisation. VEGF-A is produced by various ocular cell types in response to hypoxia and has a number of functions, including promoting vascular permeability and stimulating endothelial cell growth.
AMD is defined as an abnormality of the retinal pigment epithelium, which leads to degeneration of the overlying photoreceptor in the macula and results in loss of central vision. AMD represents a major public health burden and it is estimated that over 9 million people in the US have intermediate or advanced forms of AMD. Early AMD is characterised by drusen and hyper or hypopigmentation of the retinal pigment epithelium without loss of vision. Advanced AMD, where loss of vision occurs, can present as geographic atrophy or choriodal neovascularisation (CNV). CNV, which is also referred to as wet AMD, is a result of the abnormal growth of blood vessels.
Ranibizumab (LUCENTIS™), bevacizumab (AVASTIN™) and aflibercept (EYLEA™) are examples of anti-VEGF therapies, which are commonly administered for neovascular AMD. Despite the presence of such therapies, there exists a need for further therapies for the treatment of AMD and other ocular diseases.